DNA Repair

Question 1

relationship btwn mutations, DNA repair and cancer

Answer 1

Changes in DNA molecules can cause mutations. After replication, these changes result in a permanent alteration of the base sequences in the daughter DNA. Cancer and many human diseases are the consequence of mutation due to DNA damage and inadequate DNA repair.

Question 2

examples of heritable human diseases caused by defective DNA repair pathways

Answer 2

• Mutations in genes that mediate NER lead to human genetic diseases including Cockayne Syndrome (CS), Xeroderma pigmentosum (XP), and Trichothiodystrophy (TTD).
• Hereditary non-polyposis colorectal cancer (HNPCC) is caused by mutations in the machinery that performs mismatch repair in humans.

Question 3

DNA damage: change of base

Answer 3

• Spontaneous base loss (depurination, depyrimidination)
• Spontaneous deamination (A, C, G)
• Sunlight (UV)-induced thymidine dimers
• Base alkylation (e.g. O6-meG)
• Base oxidation by reactive oxygen species (e.g. 8-oxo-dG)

Question 4

DNA damage: change in DNA structure

Answer 4

Bulges in DNA double helix caused by:
Insertion/deletion of nucleotides Bulky chemical adducts Replication errors (mismatch) Intra/inter-strand crosslinks
• DNA strand breaks
• Stalled DNA replication forks

Question 5

Damage that can occur can cause change-causing mutations

Answer 5

oxidative damage caused
by products of normal metabolic activity of the cell, cleavage of a DNA strand caused by
radiation and chemicals, chemical alterations to the base (e.g. alkylation), loss of a base
(depurination / depyrimidination), loss of an amine group of the base (deamination), sunlight
induced thymine-dimers

Question 6

Machinery used to repair damage

Answer 6

1. direct
2. excision
3. base excision repair
4. nucleotide excision repair
5. mismatch repair
6. lesion bypass

Question 7

Direct repair

Answer 7

(ex. ligation of a break in the phosphodiester backbone of the DNA by DNA ligase or repair by MGMT)

Question 8

-Excision Repair:

Answer 8

excision of the damaged region, followed by precise replacement. Basically all excision repair pathways require endonuclease and/or exonuclease, DNA polymerase and DNA ligase.

Question 9

—Base excision repair (BER):

Answer 9

Repairs base damage that does not distort the DNA and is initiated by damage-specific glycosylases that release the damaged base, followed by removal of the damaged nucleotide.

Question 10

—Nucleotide excision repair (NER):

Answer 10

removes DNA lesions that distort the DNA structure and block RNA or DNA polymerase movement on the DNA (ex. thymidine dimers, carcinogen induced bulky additions to bases). A short oligonucleotides (13-30 nt) including the damaged base are removed via dual incision by two endonucleases. Has two modes of damage recognition

• Transcription Coupled NER, or
• Global Genome NER. Mutations in NER machinery causes CS or/ and XP conditions

Question 11

—Mismatch repair (MMR):

Answer 11

Removes nucleotides that are misincorporated during DNA replication.New strand of DNA is recognized by hemimethylation state in E. coli, and by nicks on newly synthesized DNA strand in humans.Mutations in the MMR machinery cause HNPCC

Question 12

-Lesion bypass:

Answer 12

Is used when cells have too much DNA damage for the “error-proof” repair machineries (NER, BER, MMR) to handle, especially replication-blocking lesions, and cells employ “bypass” or “error-prone” DNA polymerases with loosened specificity to enable replication to continue through damaged template strand (The bypass polymerases lack the proofreading 3’-to-5’ exonuclease activity and have error rates 100-10,000 fold higher than those of the polymerases used in normal DNA replication)

Question 13

Double stranded DNA breaks (DSBs) can be repaired by _______ or _______

Answer 13

by either non-homologous end

joining (NHEJ) or homologous recombination (HR)

Question 14

Molecular consequences of DNA damage

Answer 14

1. thymine dimers (from UV damage)
2. uracil mis-incorportations
3. bulky chemical adducts
4. double strand breaks

Question 15

Steps common to all 3 excision repair mechanism

Answer 15

1. recognition of damaged/mismatched nucleotide
2. Endonuclease-mediated cutting of phosphdiester backbone flanking the damage
3. Nuclease mediated removal of DNA fragment containing damaged/mismatched nucleotide
4. DNA poly mediated synthesis of missing dNTPS
5. DNA ligase sealing the remaining nick of phosphodiester backbone

Question 16

Steps of NER

Answer 16

1. Recognition and binding of the damaged site by a multi-protein complex (two different ways depending on local transcription activity).
2. Local unwinding of the DNA duplex by helicases (parts of the TFIIH protein complex) to form a bubble of ~25 bases.
3. Double incision of the damaged strand By two endonucleases and removal of a ~30 base oligonucleotide containing the lesion.
4. Filling in the gap by a DNA polymerase. 5. Rejoining the two ends by a DNA ligase.

Question 17

Nucleotide excision repair (NER) removes damage that

Answer 17

distorts the DNA structure and blocks poly function

Question 18

BER is initiated by

Answer 18

glycosylase, which flips out the damaged base from the stacked region of DNA duplex and removes it by hydrolyzing the glycosidic bond

Question 19

Base excision Repair removes base damage that

Answer 19

doesn’t distort DNA duplex

Question 20

BER steps

Answer 20

Step 1: Modified base is recognized by a specific DNA glycosylase, which hydrolyzes the N-glycosidic bond, yielding an AP site.
Step 2. An AP site-specific endonuclease (APE1) cleaves the sugar-phosphate backbone 5’ to the AP site.
Step 3. Another endonuclease cuts 3’ to the AP site, removing the deoxyribose phosphate.
Step 4. The resulting gap is filled by DNA polymerase, and the nick sealed by DNA ligase.

Question 21

Two ways NER recognize damage

Answer 21

Global genome NER

Txn coupled NER

Question 22

Global genome NER

Answer 22

recognize damage anywhere in genome

defects cause cancer, like XP

Question 23

Txn-coupled NER

Answer 23

recognizes damage within transcribed region

defects cause CNS disorder

cockayne sundrom (CS)

Question 24

MMR corrects errors made by

Answer 24

DNA poly during replication

Question 25

MMR is initiated by

Answer 25

two protein complexes
MutS homolog: hMSH

MutL Homologs: hMLH and PMS

Question 26

how does MMR know which strand is old vs new

Answer 26

Nascent lagging strand is marked by transient 5’ DNA ends of short discontinuous Okazaki fragments.

Nascent leading strand is marked by transient presence of ribonucleotides (1 rNMP/1,250 dNMP), which is processed into nicks by RNase H2.

Question 27

PARP: Poly(ADP-ribose) polymerase is activated by a

Answer 27

by a single-strand break and adds poly(ADP-ribose) chains to proteins.

Question 28

________ of proteins near a single strand break site facilitates DNA repair

Answer 28

Reversible Poly (ADP-ribosylation)

Question 29

PARP’s role in SSB repair:

Answer 29

1. amplification of damage signal
2. Focal enrichment of repair proteins
3. change in local chromatin structure

Question 30

DNA damage checkpoint

Answer 30

signals–> sensors–> transducers (kinases) –> effectors

Question 31

-Lesion bypass (aka translesion synthesis):

Answer 31

If a cell encounters so much DNA damage of the type that normally blocks DNA replication (such as UV-induced thymidine dimers) that the excision repair systems cannot fix it all, cells
resort to this pathway. As a last resort, cells employ “bypass” or “error-prone” DNA polymerases with loosened specificity to enable replication to continue through damaged template strand. However, it is highly mutagenic because alternate DNA polymerases that lack 3’ to 5’ proofreading exonuclease activity are used to replicate past the DNA lesion, resulting in an error rate 100-10,000 higher than normal DNA replication.

Question 32

DNA Damage Checkpoint halts

Answer 32

DNA Damage Checkpoint halts cell cycle progression when DNA is compromised to allow time for DNA repair. It is a signaling pathway composed of damage sensors, signal transducers and effectors. Protein kinases ATM and ATR are recruited to initiate the sequential recruitment and activation of downstream proteins (and delay or prevent cancer in earlier stages of tumorigenesis).

Question 33

Changes in DNA molecules can cause ___________

Answer 33

mutations

Question 34

__________ are the consequence of mutation due to DNA damage and inadequate DNA repair.

Answer 34

cancer and many human diseases

Question 35

Mutations in genes that mediate NER lead to human genetic diseases including ________ , ________, and ________.

Answer 35

Cockayne Syndrome (CS)
Xeroderma pigmentosum (XP) Trichothiodystrophy (TTD).